Search

The current study investigates the fitness cost associated with phosphine resistance in the rusty grain beetle, Cryptolestes ferrugineus (Stephens), a problematic pest in the stored commodities that has developed strong resistance to fumigant phosphine. Three characterised insect strains: the susceptible (Ref-S), the strongly resistant (Ref-R), the introgressed resistant (Intro-R) and a segregating population (F25) derived from crossing the Ref-S and Ref-R strains were used in this study. Intro-R was developed by introgressing two phosphine resistance genes, cf_rph1 and cf_rph2 into Ref-S, aimed to reduce the influence of background genetic factors. Intro-R exhibited 592 × resistance to phosphine and homozygous for strong resistance allele, cf_rph2 (L73N). Two key fitness cost criteria, developmental time and fecundity, were assessed under optimal and suboptimal conditions (less favourable diet and low temperature). There was no significant difference in developmental time and fecundity between Ref-S and either Intro-R strain or F25 under optimal conditions. When challenged with a less favourable diet, cracked wheat + cracked sorghum (CW + CS), or exposed to a low temperature (22 °C), both Intro-R and Ref-S had similar developmental time and total numbers of F1 progeny, confirming the absence of significant fitness effects expressed in these conditions. Therefore, we conclude that strongly phosphine resistant C. ferrugineus are unlikely to incur potential fitness costs. This finding will have implications towards developing strategies to manage this pest.

A decrease in energy metabolism is associated with Alzheimer's disease (AD), but it is not known whether the observed decrease exacerbates or protects against the disease. The importance of energy metabolism in AD is reinforced by the observation that variants of dihydrolipoamide dehydrogenase (DLD), is genetically linked to late-onset AD. To determine whether DLD is a suitable therapeutic target, we suppressed the dld-1 gene in Caenorhabditis elegans that express human Aβ peptide in either muscles or neurons. Suppression of the dld-1 gene resulted in significant restoration of vitality and function that had been degraded by Aβ pathology. This included protection of neurons and muscles cells. The observed decrease in proteotoxicity was associated with a decrease in the formation of toxic oligomers rather than a decrease in the abundance of the Aβ peptide. The mitochondrial uncoupler, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), which like dld-1 gene expression inhibits ATP synthesis, had no significant effect on Aβ toxicity. Proteomics data analysis revealed that beneficial effects after dld-1 suppression could be due to change in energy metabolism and activation of the pathways associated with proteasomal degradation, improved cell signaling and longevity. Thus, some features unique to dld-1 gene suppression are responsible for the therapeutic benefit. By direct genetic intervention, we have shown that acute inhibition of dld-1 gene function may be therapeutically beneficial. This result supports the hypothesis that lowering energy metabolism protects against Aβ pathogenicity and that DLD warrants further investigation as a therapeutic target.

Strong resistance to phosphine (PH3) in the rusty grain beetle, Cryptolestes ferrugineus (Stephens) (Laemophloeidae: Coleoptera) poses a serious risk to stored-grain biosecurity. Resistant populations hold risk of surviving in PH3 fumigation, particularly in storage structure that limits achieving very high concentrations of PH3, demanding the need for alternative fumigation strategies. Cofumigation with PH3 and carbon dioxide (CO2) is one alternative approach that has the potential to be used widely. CO2 fumigation of adults of strongly PH3-resistant reference strain of C. ferrugineus, for 48 h, showed that the effective concentration (LC50) of CO2 was 30.99%. This 30% level of CO2 in combination with PH3 decreased the LC50 of PH3 from 6.7 mg/liter to 0.84 mg/liter, an eightfold increase in PH3 efficacy relative to PH3 fumigation in normal air. The LC99.9 decreased from 16.2 mg/liter to 5.8 mg/liter, a 2.8-fold increase in PH3 efficacy. Comparison of mortality response data of PH3 alone and the PH3 + CO2 mixture confirmed that CO2 enhances the toxicity of PH3 synergistically in addition to exerting its own toxicity. These results were validated against three independently field-derived strains of strongly resistant C. ferrugineus that confirmed that observed enhancement in toxicity with the PH3 + CO2 mixture was consistent, irrespective of differences in resistance phenotypes and inherent tolerance levels. Results of the current study provide further opportunities to develop new commercially viable strategy to control strongly PH3-resistant C. ferrugineus.

Development of resistance in major grain insect pest species to the key fumigant phosphine (hydrogen phosphide) across the globe has put the viability and sustainability of phosphine in jeopardy. The resistance problem has been aggravated over the past two decades, due mostly to the lack of suitable alternatives matching the major attributes of phosphine, including its low price, ease of application, proven effectiveness against a broad pest spectrum, compatibility with most storage conditions, and international acceptance as a residue-free treatment. In this review, we critically analyze the published literature in the area of phosphine resistance with special emphasis on the methods available for detection of resistance, the genetic basis of resistance development, key management strategies, and research gaps that need to be addressed.

BACKGROUND: Next-generation sequencing can enable genetic surveys of large numbers of individuals. We developed a genotyping-by-sequencing assay for detecting strong phosphine resistance alleles in the dihydrolipoamide dehydrogenase (dld) gene of Rhyzopertha dominica populations. The assay can estimate the distribution and frequency of resistance variants in thousands of individual insects in a single run. RESULTS: We analysed 1435 individual insects collected over a 1-year period from 59 grain-storage sites including farms (n = 29) and central storages (n = 30) across eastern Australia. Resistance alleles were detected in 49% of samples, 38% of farms and 60% of central storages. Although multiple alleles were detected, only two resistance variants (P49S and K142E) were widespread and each appeared to have a distinct but overlapping geographical distribution. CONCLUSION: The type of structure in which the grain is stored had a strong effect on resistance allele frequency. We observed higher frequencies of resistance alleles in bunker storages at central sites compared with other storage types. This contributed to the higher frequencies of resistance alleles in bulk-handling facilities relative to farms. The discovery of a storage structure that predisposes insects to resistance highlights the utility of our high-throughput assay system for improvement of phosphine resistance management practices. © 2018 Society of Chemical Industry. © 2018 Society of Chemical Industry

Phosphine is the only fumigant approved globally for general use to control insect pests in stored grain. Due to the emergence of resistance among insect pests and the lack of suitable alternative fumigants, we are investigating ways to synergistically enhance phosphine toxicity, by studying the mechanism of action of known synergists, such as oxygen, temperature, and arsenite. Under normoxia, exposure of the model organism Caenorhabditis elegans for 24 h at 20°C to 70 ppm phosphine resulted in 10% mortality, but nearly 100% mortality if the oxygen concentration was increased to 80%. In wild-type C. elegans, toxicity of phosphine was negatively affected by a decrease in temperature to 15°C and positively affected by an increase in temperature to 25°C. The dld-1(wr4) strain of C. elegans is resistant to phosphine due to a mutation in the dihydrolipoamide dehydrogenase gene. It also exhibits increased mortality that is dependent on hyperoxia, when exposed to 70 ppm phosphine at 20°C. As with the wild-type strain, mortality decreased when exposure was carried out at 15°C. At 25°C, however, the strain was completely resistant to the phosphine exposure at all oxygen concentrations. Arsenite is also a synergist of phosphine toxicity, but only in the dld-1(wr4)-mutant strain. Thus, exposure to 4 mM arsenite resulted in 50% mortality, which increased to 89% mortality when 70 ppm phosphine and 4 mM arsenite were combined. In stark contrast, 70 ppm phosphine rendered 4 mM arsenite nontoxic to wild-type C. elegans. These results reveal two synergists with distinct modes of action, one of which targets individuals that carry a phosphine resistance allele in the dihydrolipoamide dehydrogenase gene.

Dung beetles are a very important part of an ecosystem because of their role in the removal and decomposition of vertebrate dung. It has been suspected that symbiotic gut bacteria facilitate this role, a hypothesis that we have explored with high-throughput barcoding., Gut microbes play an important role in the biology and evolution of insects. Australian native dung beetles (Scarabaeinae) present an opportunity to study gut microbiota in an evolutionary context as they come from two distinct phylogenetic lineages and some species in each lineage have secondarily adapted to alternative or broader diets. In this study, we characterized the hindgut bacterial communities found in 21 species of dung beetles across two lineages, using 16S rRNA sequencing. We found that gut microbial diversity was more dependent on host phylogeny and gut morphology than specific dietary preferences or environment. In particular, gut microbial diversity was highest in the endemic, flightless genus Cephalodesmius, which feeds on a broad range of composted organic matter. The hindgut of Cephalodesmius beetles harbors a highly conserved core set of bacteria, suggesting that the bacteria are symbiotic. Symbiosis is supported by the persistence of the core microbiota across isolated beetle populations and between species in the genus. A coevolutionary relationship is supported by the expansion of the hindgut to form a fermentation chamber and the fermentative nature of the core microbes. In contrast, Australian species of the widespread dung beetle genus Onthophagus that specialize on a single food resource, such as dung or fungus, exhibit minimal food processing behavior and have a short, narrow hindgut and a variable gut microbiota with relatively few core bacterial taxa. A conserved, complex gut microbiota is hypothesized to be unnecessary for this highly mobile genus. IMPORTANCE Dung beetles are a very important part of an ecosystem because of their role in the removal and decomposition of vertebrate dung. It has been suspected that symbiotic gut bacteria facilitate this role, a hypothesis that we have explored with high-throughput barcoding. We found that differences in hindgut morphology had the greatest effect on the bacterial community composition. Species with a hindgut fermentation chamber harbored a distinctly different hindgut community compared to those species with a narrow, undifferentiated hindgut. Diet and phylogeny were also associated with differences in gut community. Further understanding of the relationships between dung beetles and their gut microbes will provide insights into the evolution of their behaviors and how gut communities contribute to their fitness.

Alzheimer's disease (AD) is an age-related disease characterized by loss of memory and disrupted thinking that is associated with altered energy metabolism. Variants of an important enzyme of energy metabolism, dihydrolipoamide dehydrogenase (dld), have been genetically linked to late-onset AD. Moreover, reduced activity of DLD-containing enzyme complexes is associated with AD progression. To understand how energy metabolism influences AD progression, we exposed C. elegans expressing human Aβ peptide to the chemical inhibitor of DLD, 2-methoxyindole-5-carboxylic acid (MICA). Expression of human Aβ in C. elegans causes a variety of pathologies that can be used to monitor the efficacy of treatments against proteotixicity. We found that MICA alleviated the Aβ-induced paralysis and improved cholinergic neurotransmission in C. elegans that express Aβ in muscle cells. MICA also reduced both hypersensitivity to serotonin and perturbation of chemotaxis associated with neuronal expression of human Aβ. Furthermore, low doses of MICA helped to alleviate an Aβ-mediated decrease in fecundity. Protection against AD pathogenesis by MICA in the C. elegans model was associated with a decrease in Aβ oligomerization that could be reversed by the calcium ionophore, A23187. MICA also caused a decrease in oxidative stress, which could also contribute to the protective effect of MICA against Aβ toxicity.

Although phosphine resistance monitoring and management programs are well advanced in Australia, the tropical northern part of the continent has received little attention. To address this, grain storages were sampled in the Townsville region in the Burdekin river catchment area of northern Queensland, and insect populations were subjected to resistance testing. Sampling of storages including silos, food processing facilities and feedlots was carried out during July to August 2019 and February to March 2020. Of the several species collected, populations of two major pest species, Rhyzopertha dominica and Tribolium castaneum were subjected to phenotypic resistance and molecular screening using discriminating doses and gene specific DNA marker, respectively. Utilising phenotypic assay, of the 17 populations of R. dominica, 12 were diagnosed as weakly resistant and five as strongly resistant; whereas of the 34 populations of T. castaneum, two were found to be susceptible to phosphine, 15 were determined as weakly resistant and 17 as strongly resistant. The molecular diagnostics, however, determined rph2 alleles for strong resistance in some populations each of R. dominica and T. castaneum that were diagnosed as weakly resistant in the phenotypic assay. The most prevalent rph2 alleles were found to be P49S and K142E in R. dominica populations and P45S and G131S in T. castaneum populations. Our results highlight the need of more comprehensive study towards determining the level of pests and resistance risks in this region.

One of the loci responsible for strong phosphine resistance encodes dihydrolipoamide dehydrogenase (DLD). The strong co-incidence of enzyme complexes that contain DLD, and enzymes that require thiamine as a cofactor, motivated us to test whether the thiamine deficiency of polished white rice could influence the efficacy of phosphine fumigation against insect pests of stored grain. Three strains of Sitophilus oryzae (susceptible, weak and strong resistance) were cultured on white rice (thiamine deficient), brown rice or whole wheat. As thiamine is an essential nutrient, we firstly evaluated the effect of white rice on developmental rate and fecundity and found that both were detrimentally affected by this diet. The mean time to reach adult stage for the three strains ranged from 40 to 43 days on brown rice and 50–52 days on white rice. The mean number of offspring for the three strains ranged from 7.7 to 10.3 per female over a three day period on brown rice and 2.1 to 2.6 on white rice. Growth and reproduction on wheat was similar to that on brown rice except that the strongly resistant strain showed a tendency toward reduced fecundity on wheat. The susceptible strain exhibited a modest increase in tolerance to phosphine on white rice as expected if thiamine deficiency could mimic the effect of the dld resistance mutation at the rph2 locus. The strongly resistant strain did not respond to thiamine deficiency, but this was expected as these insects are already strongly resistant. We failed, however, to observe the expected synergistic increase in resistance due to combining thiamine deficiency with the weakly resistant strain. The lack of interaction between thiamine content of the diet and the resistance genotype in determining the phosphine resistance phenotype suggests that the mode of inhibition of the complexes is a critical determinant of resistance.

Phosphine gas is an excellent fumigant for disinfesting stored grain of insect pests, but heavy reliance on phosphine has led to resistance in grain pests that threatens its efficacy. Phosphine-resistance was previously reported to be mediated by the enzyme DLD. Here we explore the relationship between phosphine toxicity and genotoxic treatments with the goal of understanding how phosphine works. Specifically, we utilized mutant lines either sensitive or resistant to phosphine, gamma irradiation or UV exposure. The phosphine-resistance mutation in the enzyme of energy metabolism, dihydrolipoamide dehydrogenase exhibited cross-resistance to UV and ionizing radiation. Two radiation-sensitive mutants that are defective in DNA repair as well as a mutant that is defective in the activation of the DAF-16 stress response transcription factor all exhibit sensitivity to phosphine that exceeds the sensitivity of the wild type control. A radiation resistance mutation in cep-1, the p53 orthologue, that is deficient in double strand break repair of DNA and is also deficient in apoptosis causes radiation-resistance results but sensitivity toward phosphine.

The rusty grain beetle, Cryptolestes ferrugineus, a major pest of stored commodities, has developed very high levels (>1000×) of resistance to the fumigant phosphine. Resistance in this species is remarkably stronger than reported in any other stored product pests demanding the need to understand the molecular basis of this trait. Previous genetic studies in other grain insect pests identified specific variants in two major genes, rph1 and rph2 in conferring the strong resistance trait. However, in C. ferrugineus, although the gene, rph1 was identified as cytochrome-b5-fatty acid desaturase, the rph2 gene has not been reported so far. We tested the candidate gene for rph2, dihydrolipoamide dehydrogenase (dld) using the recently published transcriptome of C. ferrugineus and identified three variants, L73N and A355G + D360H, a haplotype, conferring resistance in this species. Our sequence analysis in resistant strain and phosphine selected resistant survivors indicates that these variants occur either alone as a homozygote or a mixture of heterozygotes (i.e complex heterozygotes) both conferring strong resistance. We also found that one of the three variants, possibly L73N expressing “dominant” trait at low frequency in resistant insects. Comparison of dld sequences between Australian and Chinese resistant strain of this species confirmed that the identified variants are highly conserved. Our fitness analysis indicated that resistant insects may not incur significant biological costs in the absence of phosphine selection for 19 generations. Thus, we propose that the observed high levels of resistance in C. ferrugineus could be primarily due to the characteristics of three unique variants, L73N and A355G + D360H within dld.

Inheritance of resistance to phosphine fumigant was investigated in three field-collected strains of rusty grain beetle, Cryptolestes ferrugineus, Susceptible (S-strain), Weakly Resistant (Weak-R) and Strongly Resistant (Strong-R). The strains were purified for susceptibility, weak resistance and strong resistance to phosphine, respectively, to ensure homozygosity of resistance genotype. Crosses were established between S-strain×Weak-R, S-strain×Strong-R and Weak-R×Strong-R, and the dose mortality responses to phosphine of these strains and their F1, F2 and F1-backcross progeny were obtained. The fumigations were undertaken at 25°C and 55% RH for 72h. Weak-R and Strong-R showed resistance factors of 6.3× and 505× compared with S-strain at the LC50. Both weak and strong resistances were expressed as incompletely recessive with degrees of dominance of -0.48 and -0.43 at the LC50, respectively. Responses of F2 and F1-backcross progeny indicated the existence of one major gene in Weak-R, and at least two major genes in Strong-R, one of which was allelic with the major factor in Weak-R. Phenotypic variance analyses also estimated that the number of independently segregating genes conferring weak resistance was 1 (nE=0.89) whereas there were two genes controlling strong resistance (nE=1.2). The second gene, unique to Strong-R, interacted synergistically with the first gene to confer a very high level of resistance (~80×). Neither of the two major resistance genes was sex linked. Despite the similarity of the genetics of resistance to that previously observed in other pest species, a significant proportion (~15 to 30%) of F1 individuals survived at phosphine concentrations higher than predicted. Thus it is likely that additional dominant heritable factors, present in some individuals in the population, also influenced the resistance phenotype. Our results will help in understanding the process of selection for phosphine resistance in the field which will inform resistance management strategies. In addition, this information will provide a basis for the identification of the resistance genes.

Resistance to phosphine (PH3) in key insect pests of stored grain is increasing, with a requirement for maintaining a dose as high as 1 mg l-1 for 14 d for effective fumigation, which is difficult to achieve under most commercial storage conditions. There is no suitable replacement for PH3, as most of the available alternatives suffer from specific weaknesses, creating an urgent need to increase the efficacy of this fumigant. One such possibility is co-fumigation of PH3 with another fumigant, sulfuryl fluoride (SO2F2-SF), with the goal of decreasing the time required for a successful fumigation. In this study, adult of two PH3-resistant strains in each of four key grain insect pests, Rhyzopertha dominica, (F.) (Coleoptera: Bostrichidae) Tribolium castaneum Herbst (Coleoptera: Tenebrionidae), Sitophilus oryzae (L.) (Coleoptera: Curculionidae), and Cryptolestes ferrugineus (Stephens) (Coleoptera: Laemophloeidae) were fumigated with PH3 and SF individually as well as in combinations at 25°C over 48 h. Mortality responses in each species were subjected to probit analysis to determine the LC50 and LC99.9 of PH3, SF, and PH3 + SF. Co-fumigation of PH3 with SF resulted in a 50% reduction of the PH3 concentration required to achieve 99.9% mortality in two pest species. For example, the PH3 + SF mixture, reduced the amount of PH3 required from 14.2-14.5 to 5.6-6.36 mg l-1 and from 2.71-5.03 to 0.93-1.2 mg l-1, respectively, for C. ferrugineus and S. oryzae. The overall mortality response to the PH3 + SF mixture followed an "additive model" suggesting that mutual enhancement in toxicity can be achieved with this mixture specifically to control PH3-resistant insects.

Next-generation sequencing methods enable identification of the genetic basis of traits in species that have no prior genomic information available. The combination of next-generation sequencing, variant analysis, and linkage is a powerful way of identifying candidate genes for a trait of interest. Here, we used a comparative transcriptomics [RNA sequencing (RNAseq)] and genetic linkage analysis approach to identify the rph1 gene. rph1 variants are responsible for resistance to the fumigant phosphine (PH3) that is used to control insect pests of stored grain. In each of the four major species of pest insect of grain we have investigated, there are two major resistance genes, rph1 and rph2, which interact synergistically to produce strongly phosphine-resistant insects. Using RNAseq and genetic linkage analyses, we identified candidate resistance (rph1) genes in phosphine-resistant strains of three species: Rhyzopertha dominica (129 candidates), Sitophilus oryzae (206 candidates), and Cryptolestes ferrugineus (645 candidates). We then compared these candidate genes to 17 candidate resistance genes previously mapped in Tribolium castaneum and found only one orthologous gene, a cytochrome b5 fatty acid desaturase (Cyt-b5-r), to be associated with the rph1 locus in all four species. This gene had either missense amino acid substitutions and/or insertion/deletions/frameshift variants in each of 18 phosphine-resistant strains that were not observed in the susceptible strains of the four species. We propose a model of phosphine action and resistance in which phosphine induces lipid peroxidation through reactive oxygen species generated by dihydrolipoamide dehydrogenase, whereas disruption of Cyt-b5-r in resistant insects decreases the polyunsaturated fatty acid content of membranes, thereby limiting the potential for lipid peroxidation.

Phosphine is the only general use fumigant for the protection of stored grain, though its long-term utility is threatened by the emergence of highly phosphine-resistant pests. Given this precarious situation, it is essential to identify factors, such as stress preconditioning, that interfere with the efficacy of phosphine fumigation. We used Caenorhabditis elegans as a model organism to test the effect of pre-exposure to heat and cold shock, UV and gamma irradiation on phosphine potency. Heat shock significantly increased tolerance to phosphine by 3-fold in wild-type nematodes, a process that was dependent on the master regulator of the heat shock response, HSF-1. Heat shock did not, however, increase the resistance of a strain carrying the phosphine resistance mutation, dld-1(wr4), and cold shock did not alter the response to phosphine of either strain. Pretreatment with the LD50 of UV (18 J cm-2) did not alter phosphine tolerance in wild-type nematodes, but the LD50 (33 J cm-2) of the phosphine resistant strain (dld-1(wr4)) doubled the level of resistance. In addition, exposure to a mild dose of gamma radiation (200 Gy) elevated the phosphine tolerance by ~2-fold in both strains.

Phosphine (PH3) fumigation is the primary method worldwide for controlling insect pests of stored commodities. Over-reliance on phosphine, however, has led to the emergence of strong resistance. Detailed genetic studies previously identified two loci, rph1 and rph2, that interact synergistically to create a strong resistance phenotype. We compared the genetics of phosphine resistance in strains of Rhyzopertha dominica and Tribolium castaneum from India and Australia, countries having similar pest species but widely differing in pest management practices. Sequencing analysis of the rph2 locus, dihydrolipoamide dehydrogenase (dld), identified two structurally equivalent variants, Proline49>Serine (P49S) in one R. dominica strain and P45S in three strains of T. castaneum from India. These variants of the DLD protein likely affect FAD cofactor interaction with the enzyme. A survey of insects from storage facilities across southern India revealed that the P45/49S variant is distributed throughout the region at very high frequencies, in up to 94% of R. dominica and 97% of T. castaneum in the state of Tamil Nadu. The abundance of the P45/49S variant in insect populations contrasted sharply with the evolutionary record in which the variant was absent from eukaryotic DLD sequences. This suggests that the variant is unlikely to provide a strong selective advantage in the absence of phosphine fumigation.

BackgroundPest insects are increasingly resistant to phosphine gas, which is used globally to protect grain reserves. The enzyme dihydrolipoamide dehydrogenase (DLD) is a phosphine resistance factor and participates in four key steps of core metabolism, making it a potential central metabolic regulator.ResultsHere we used microarray data and NMR-based metabolomics to characterize the phosphine response of wild-typeC. elegansand the phosphine-resistant straindld-1(wr4) which has a partial loss-of-function mutation in the gene for DLD. In addition, we have constructedCeCon, aC. elegansgenome-scale metabolic model to facilitate integration of gene expression and metabolomics data.ConclusionsThe resulting systems biology analysis is consistent with the hypothesis that adaptation to a hypometabolic state is the most prominent mechanism of phosphine resistance in this nematode strain. The involvement of DLD in regulating and creating hypometabolic adaptation has implications for other biological phenomena involving hypometabolism, such as reperfusion injury and metabolic resistance.

Phosphine resistance alleles might be expected to negatively affect energy demanding activities such as walking and flying, because of the inverse relationship between phosphine resistance and respiration. We used an activity monitoring system to quantify walking of Rhyzopertha dominica (F.) and a flight chamber to estimate their propensity for flight initiation. No significant difference in the duration of walking was observed between the strongly resistant, weakly resistant, and susceptible strains of R. dominica we tested, and females walked significantly more than males regardless of genotype. The walking activity monitor revealed no pattern of movement across the day and no particular time of peak activity despite reports of peak activity of R. dominica and Tribolium castaneum (Herbst) under field conditions during dawn and dusk. Flight initiation was significantly higher for all strains at 28 degrees C and 55% relative humidity than at 25, 30, 32, and 35 degrees C in the first 24 h of placing beetles in the flight chamber. Food deprivation and genotype had no significant effect on flight initiation. Our results suggest that known resistance alleles in R. dominica do not affect insect mobility and should therefore not inhibit the dispersal of resistant insects in the field.

BACKGROUND: The lesser grain borer, Rhyzopertha dominica (F.), is a highly destructive pest of stored grain that is strongly resistant to the fumigant phosphine (PH3). Phosphine resistance is due to genetic variants at the rph2 locus that alter the function of the dihydrolipoamide dehydrogenase (DLD) gene. This discovery now enables direct detection of resistance variants at the rph2 locus in field populations. RESULTS: A genotype assay was developed for direct detection of changes in distribution and frequency of a phosphine resistance allele in field populations of R. dominica. Beetles were collected from ten farms in south-east Queensland in 2006 and resampled in 2011. Resistance allele frequency increased in the period from 2006 to 2011 on organic farms with no history of phosphine use, implying that migration of phosphine-resistant R. dominica had occurred from nearby storages. CONCLUSION: Increasing resistance allele frequencies on organic farms suggest local movement of beetles and dispersal of insects from areas where phosphine has been used. This research also highlighted for the first time the utility of a genetic DNA marker in accurate and rapid determination of the distribution of phosphine-resistant insects in the grain value chain. Extending this research over larger landscapes would help in identifying resistance problems and enable timely pest management decisions. © 2013 Society of Chemical Industry © 2013 Society of Chemical Industry 69 6 June 2013 10.1002/ps.3514 Rapid Report Rapid Report © 2013 Society of Chemical Industry.

Dissecting Phosphine Resistance Worldwide populations of pest insects—such as the lesser grain borer, Rhyzopertha dominica , and the rust-red flour beetle, Tribolium castaneum —have become highly resistant to the fumigant phosphine, providing a potential threat to global food security. The nematode, Caenorhabditis elegans is vulnerable to phosphine, but phosphine-resistant strains are known. Schlipalius et al. (p. 807 ) show that mutations in the delta-1-pyrroline-5-carboxylate dehydrogenase and dihydrolipoamide dehydrogenase ( dld-1 ) genes both give rise to phosphine resistance in C. elegans . Phosphine resistance mutants in R. dominica , and T. castaneum also map to the dld-1 gene, which codes for a core metabolic enzyme. These mutants are, however, hypersensitive to arsenic, mimics of which might thus synergize with phosphine.

Fumigation of stored grain with phosphine (PH 3) is used widely to control the lesser grain borer Rhyzopertha dominica. However, development of high level resistance to phosphine in this species threatens control. Effective resistance management relies on knowledge of the expression of resistance in relation to dosage at all life stages. Therefore, we determined the mode of inheritance of phosphine resistance and strength of the resistance phenotype at each developmental stage. We achieved this by comparing mortality and developmental delay between a strongly resistant strain (R-strain), a susceptible strain (S-strain) and their F 1 progenies. Resistance was a maternally inherited, semi-dominant trait in the egg stage but was inherited as an autosomal, incompletely recessive trait in larvae and pupae. The rank order of developmental tolerance in both the sensitive and resistant strains was eggs > pupae > larvae. Comparison of published values for the response of adult R. dominica relative to our results from immature stages reveals that the adult stage of the S-strain is more sensitive to phosphine than are larvae. This situation is reversed in the R-strain as the adult stage is much more resistant to phosphine than even the most tolerant immature stage. Phosphine resistance factors at LC 50 were eggs 400×, larvae 87× and pupae 181× with respect to reference susceptible strain (S-strain) adults indicating that tolerance conferred by a particular immature stage neither strongly nor reliably interacts with the genetic resistance element. Developmental delay relative to unfumigated control insects was observed in 93% of resistant pupae, 86% of resistant larvae and 41% of resistant eggs. Increased delay in development and the toxicity response to phosphine exposure were both incompletely recessive. We show that resistance to phosphine has pleiotropic effects and that the expression of these effects varies with genotype and throughout the life history of the insect. © 2012.

High levels of resistance to phosphine in the rice weevil Sitophilus oryzae have been detected in Asian countries including China and Vietnam, however there is limited knowledge of the genetic mechanism of resistance in these strains. We find that the genetic basis of strong phosphine resistance is conserved between strains of S. oryzae from China, Vietnam, and Australia. Each of 4 strongly resistant strains has an identical amino acid variant in the encoded dihydrolipoamide dehydrogenase (DLD) enzyme that was previously identified as a resistance factor in Rhyzopertha dominica and Tribolium castaneum. The unique amino acid substitution, Asparagine > Threonine (N505T) of all strongly resistant S. oryzae corresponds to the position of an Asparagine > Histidine variant (N506H) that was previously reported in strongly resistant R. dominica. Progeny (F16 and F18) from 2 independent crosses showed absolute linkage of N505T to the strong resistance phenotype, indicating that if N505T was not itself the resistance variant that it resided within 1 or 2 genes of the resistance factor. Non-complementation between the strains confirmed the shared genetic basis of strong resistance, which was supported by the very similar level of resistance between the strains, with LC50 values ranging from 0.20 to 0.36 mg L(-1) for a 48-h exposure at 25 °C. Thus, the mechanism of high-level resistance to phosphine is strongly conserved between R. dominica, T. castaneum and S. oryzae. A fitness cost associated with strongly resistant allele was observed in segregating populations in the absence of selection.

Oxidative stress is a toxic state caused by an imbalance between the production and elimination of reactive oxygen species (ROS). ROS cause oxidative damage to cellular components such as proteins, lipids, and nucleic acids. While the role of ROS in cellular damage is frequently all that is noted, ROS are also important in redox signalling. The “Redox Hypothesis" has been proposed to emphasize a dual role of ROS. This hypothesis suggests that the primary effect of changes to the redox state is modified cellular signalling rather than simply oxidative damage. In extreme cases, alteration of redox signalling can contribute to the toxicity of ROS, as well as to ageing and age-related diseases. The nematode speciesCaenorhabditis elegansprovides an excellent model for the study of oxidative stress and redox signalling in animals. We use protein sequences from central redox systems inHomo sapiens,Drosophila melanogaster, andSaccharomyces cerevisiaeto query Genbank for homologous proteins inC. elegans. We then use maximum likelihood phylogenetic analysis to compare protein families betweenC. elegansand the other organisms to facilitate future research into the genetics of redox biology.

Phosphine, a widely used fumigant for the protection of stored grain from insect pests, kills organisms indirectly by inducing oxidative stress. High levels of heritable resistance to phosphine in the insect pest of stored grain, Rhyzopertha dominica have been detected in Asia, Australia and South America. In order to understand the evolution of phosphine resistance and to isolate the responsible genes, we have undertaken genetic linkage analysis of fully sensitive (QRD14), moderately resistant (QRD369) and highly resistant (QRD569) strains of R. dominica collected in Australia. We previously determined that two loci, rph1 and rph2, confer high-level resistance on strain QRD569, which was collected in 1997. We have now confirmed that rph1 is responsible for the moderate resistance of strain QRD369, which was collected in 1990, and is shared with a highly resistant strain from the same geographical region, QRD569. In contrast, rph2 by itself confers only very weak resistance, either as a heterozygote or as a homozygote and was not discovered in the field until weak resistance (probably due to rph1) had become ubiquitous. Thus, high-level resistance against phosphine has evolved via stepwise acquisition of resistance alleles, first at rph1 and thereafter at rph2. The semi-dominance of rph2 together with the synergistic interaction between rph1 and rph2 would have led to rapid selection for homozygosity. A lack of visible fitness cost associated with alleles at either locus suggests that the resistance phenotype will persist in the field.

Depolanmis hububattaki zararlilarla mucadeledeki ana yontem fosfin gazi ile fumigasyondur. Fosfin gazi Turkiye’de 1950’li yillardan beri yogun olarak kullanilmaktadir. Fosfinin uzun yillardan beri depolanmis hububatta kullanimina ve dunyada da pek cok ulkede yuksek seviyelerde fosfin direncinin belirlenmis olmasina ragmen Turkiye’de bugune kadar bu konuda herhangi bir calisma yapilmamistir. Yuksek seviyelerde fosfin direnci, un biti Tribolium castaneum ’da belirlenerek direnc yapisi genetik olarak tanimlanmistir. Bu zararli Turkiye’de depolanmis bugdayda yaygin bir zararli konumunda oldugundan, bu ilk calisma fosfin direncinin T. castaneum’ daki durumunu ve ulkedeki dagilimini ortaya koymak uzere yurutulmustur. Dort populasyon, zayif ve kuvvetli direnc icin sirasiyla ayirici fosfin konsantrasyonlari olan 0.03mg/L ve 0.25mg/L dozlari ile 20 saat sureyle uygulama temeline dayanan bioassaylerle test edilmislerdir. Bu testlerde populasyonlarin tumunde direnc oldugu ve direnc oraninin 196 kata kadar ulastigi belirlenmistir. Yapilan genetik calismalar ve sekans, tum populasyonlarin kuvvetli direnc allellerine sahip olduklarini ve bu populasyonlarin daha once literaturde rapor edilenlere benzer olarak rph2 lokusundaki direnc allelini tasidiklarini gostermistir. Calisma sonuclarimiz, T. castaneum ’un Turkiye populasyonlarinda fosfine karsi guclu bir direncin yaygin oldugunu gostermektedir.

Sitophilus oryzae (Linnaeus) is a major pest of stored grain across Southeast Asia and is of increasing concern in other regions due to the advent of strong resistance to phosphine, the fumigant used to protect stored grain from pest insects. We investigated the inheritance of genes controlling resistance to phosphine in a strongly resistant S. oryzae strain (NNSO7525) collected in Australia and find that the trait is autosomally inherited and incompletely recessive with a degree of dominance of -0.66. The strongly resistant strain has an LC50 52 times greater than a susceptible reference strain (LS2) and 9 times greater than a weakly resistant strain (QSO335). Analysis of F2 and backcross progeny indicates that two or more genes are responsible for strong resistance, and that one of these genes, designated So_rph1, not only contributes to strong resistance, but is also responsible for the weak resistance phenotype of strain QSO335. These results demonstrate that the genetic mechanism of phosphine resistance in S. oryzae is similar to that of other stored product insect pests. A unique observation is that a subset of the progeny of an F1 backcross generation are more strongly resistant to phosphine than the parental strongly resistant strain, which may be caused by multiple alleles of one of the resistance genes.

Phosphine is the primary fumigant used to protect the majority of the world' s grain and a variety of other stored commodities from insect pests. Phosphine is playing an increasingly important role in the protection of commodities for two primary reasons. Firstly, use of the alternative fumigant, methyl bromide, has been sharply curtailed and is tightly regulated due to its role in ozone depletion, and secondly, consumers are becoming increasingly intolerant of contact pesticides. Niche alternatives to phosphine exist, but they suffer from a range of factors that limit their use, including: 1) Limited commercial adoption due to expense or slow mode of action; 2) Poor efficacy due to low toxicity, rapid sorption, limited volatility or high density; 3) Public health concerns due to toxicity to handlers or nearby residents, as well as risk of explosion; 4) Poor consumer acceptance due to toxic residues or smell. These same factors limit the prospects of quickly identifying and deploying a new fumigant. Given that resistance toward phosphine is increasing among insect pests, improved monitoring and management of resistance is a priority. Knowledge of the mode of action of phosphine as well as the mechanisms of resistance may also greatly reduce the effort and expense of identifying synergists or novel replacement compounds.

The promoter regions of plant pararetroviruses direct transcription of the full-length viral genome into a pregenomic RNA that is an intermediate in the replication of the virus. It serves as template for reverse transcription and as polycistronic mRNA for translation to viral proteins. We have identified functional promoter elements in the intergenic region of the Cavendish isolate of Banana streak virus (BSV-Cav), a member of the genus Badnavirus. Potential binding sites for plant transcription factors were found both upstream and downstream of the transcription start site by homology search in the PLACE database of plant cis-acting elements. The functionality of these putative cis-acting elements was tested by constructing loss-of-function and regain-of-function mutant promoters whose activity was quantified in embryogenic sugarcane suspension cells. Four regions that are important for activity of the BSV-Cav promoter were identified: the region containing an as-l-like element, the region around-141 and down to -77, containing several putative transcription factor binding sites, the region including the CAAT-box, and the leader region. The results could help explain the high BSV-Cav promoter activity that was observed previously in transgenic sugarcane plants and give more insight into the plant cell-mediated replication of the viral genome in banana streak disease. (C) 2004 Elsevier B.V. All rights reserved.

Biogenic amines and their receptors regulate and modulate many physiological and behavioural processes in animals. In vertebrates, octopamine is only found in trace amounts and its function as a true neurotransmitter is unclear. In protostomes, however, octopamine can act as neurotransmitter, neuromodulator and neurohormone. In the honeybee, octopamine acts as a neuromodulator and is involved in learning and memory formation. The identification of potential octopamine receptors is decisive for an understanding of the cellular pathways involved in mediating the effects of octopamine. Here we report the cloning and functional characterization of the first octopamine receptor from the honeybee, Apis mellifera. The gene was isolated from a brain-specific cDNA library. It encodes a protein most closely related to octopamine receptors from Drosophila melanogaster and Lymnea stagnalis. Signalling properties of the cloned receptor were studied in transiently transfected human embryonic kidney (HEK) 293 cells. Nanomolar to micromolar concentrations of octopamine induced oscillatory increases in the intracellular Ca2+ concentration. In contrast to octopamine, tyramine only elicited Ca2+ responses at micromolar concentrations. The gene is abundantly expressed in many somata of the honeybee brain, suggesting that this octopamine receptor is involved in the processing of sensory inputs, antennal motor outputs and higher-order brain functions.

Dopamine is found in many invertebrate organisms, including insects, however, the mechanisms through which this amine operates remain unclear. We have expressed two dopamine receptors cloned from honey bee (AmDOP1 and AmDOP2) in insect cells (Spodoptera frugiperda), and compared their pharmacology directly using production of cAMP as a functional assay. In each assay, AmDOP1 receptors required lower concentrations of dopamine and 6,7-ADTN for maximal activation than AmDOP2 receptors. Conversely, butaclamol and cis(Z)-flupentixol were more potent at blocking the cAMP response mediated through AmDOP2 than AmDOP1 receptors. Expression of AmDOP1, but not AmDOP2, receptors significantly increased levels of cAMP even in the absence of ligand. This constitutive activity was blocked by cis(Z)-flupentixol. This work provides the first evidence of a constitutively activated dopamine receptor in invertebrates and suggests that although AmDOP1 and AmDOP2 share much less homology than their vertebrate counterparts, they display a number of functional parallels with the mammalian D1-like dopamine receptors.

Chemosensory proteins (CSPs) are ubiquitous soluble small proteins isolated from sensory organs of a wide range of insect species, which are believed to be involved in chemical communication. We report the cloning of a honeybee CSP gene called ASP3c, as well as the structural and functional characterization of the encoded protein. The protein was heterologously secreted by the yeast Pichia pastoris using the native signal peptide. ASP3c disulfide bonds were assigned after trypsinolysis followed by chromatography and mass spectrometry combined with microsequencing. The pairing (Cys(I)-Cys(II), Cys(III)-Cys(IV)) was found to be identical to that of Schistocerca gregaria CSPs, suggesting that this pattern occurs commonly throughout the insect CSPs. CD measurements revealed that ASP3c mainly consists of alpha-helices, like other insect CSPs. Gel filtration analysis showed that ASP3c is monomeric at neutral pH. Using ASA, a fluorescent fatty acid anthroyloxy analogue as a probe, ASP3c was shown to bind specifically to large fatty acids and ester derivatives, which are brood pheromone components, in the micromolar range. It was unable to bind tested general odorants and other tested pheromones (sexual and nonsexual). This is the first report on a natural pheromonal ligand bound by a recombinant CSP with a measured affinity constant.

High levels of inheritable resistance to phosphine in Rhyzopertha dominica have recently been detected in Australia and in an effort to isolate the genes responsible for resistance we have used random amplified DNA fingerprinting (RAF) to produce a genetic linkage map of R. dominica. The map consists of 94 dominant DNA markers with an average distance between markers of 4.6 cM and defines nine linkage groups with a total recombination distance of 390.1 cM. We have identified two loci that are responsible for high-level resistance. One provides ∼50× resistance to phosphine while the other provides 12.5× resistance and in combination, the two genes act synergistically to provide a resistance level 250× greater than that of fully susceptible beetles. The haploid genome size has been determined to be 4.76 × 108 bp, resulting in an average physical distance of 1.2 Mbp per map unit. No recombination has been observed between either of the two resistance loci and their adjacent DNA markers in a population of 44 fully resistant F5 individuals, which indicates that the genes are likely to reside within 0.91 cM (1.1 Mbp) of the DNA markers.

A GENiE (EU COST action, www.worm-genie.eu) workshop was held at the Babraham Institute in Cambridge, UK on April 19 and 20, 2017, to discuss global challenges around Caenorhabditis elegans metabol...

Existing procedures for the generation of polymorphic DNA markers are not optimal for insect studies in which the organisms are often tiny and background molecular information is often non-existent. We have used a new high throughput DNA marker generation protocol called randomly amplified DNA fingerprints (RAF) to analyse the genetic variability in three separate strains of the stored grain pest, Rhyzopertha dominica. This protocol is quick, robust and reliable even though it requires minimal sample preparation, minute amounts of DNA and no prior molecular analysis of the organism. Arbitrarily selected oligonucleotide primers routinely produced approximately 50 scoreable polymorphic DNA markers, between individuals of three independent field isolates of R. dominica. Multivariate cluster analysis using forty-nine arbitrarily selected polymorphisms generated from a single primer reliably separated individuals into three clades corresponding to their geographical origin. The resulting clades were quite distinct, with an average genetic difference of 37.5 +/- 6.0% between clades and of 21.0 +/- 7.1% between individuals within clades. As a prelude to future gene mapping efforts, we have also assessed the performance of RAF under conditions commonly used in gene mapping. In this analysis, fingerprints from pooled DNA samples accurately and reproducibly reflected RAF profiles obtained from individual DNA samples that had been combined to create the bulked samples.

In the honeybee the cAMP-dependent signal transduction cascade has been implicated in processes underlying learning and memory, The cAMP-dependent protein kinase (PKA) is the major mediator of cAMP action. To characterize the PKA system in the honeybee brain we cloned a homologue of a PKA catalytic subunit from the honeybee,The deduced amino acid sequence shows 80-94% identity with catalytic subunits of PKA from Drosophila melanogaster, Aplysia californica and mammals. The corresponding gene is predominantly expressed in the mushroom bodies, a structure that is involved in learning and memory processes. However, expression can also be found in the antennal and optic lobes,The level of expression varies within all three neuropiles.

Two putative promoters from Australian banana streak badnavirus (BSV) isolates were analysed for activity in different plant species. In transient expression systems the My (2105 bp) and Cv (1322 bp) fragments were both shown to have promoter activity in a wide range of plant species including monocots (maize, barley, banana, millet, wheat, sorghum), dicots (tobacco, canola, sunflower, Nicotiana benthamiana, tipu tree), gymnosperm (Pinus radiata) and fern (Nephrolepis cordifolia). Evaluation of the My and Cv promoters in transgenic sugarcane, banana and tobacco plants demonstrated that these promoters could drive high-level expression of either the green fluorescent protein (GFP) or the beta-glucuronidase (GUS) reporter gene (uidA) in vegetative plant cells. In transgenic sugarcane plants harbouring the Cv promoter, GFP expression levels were comparable or higher (up to 1.06% of total soluble leaf protein as GFP) than those of plants containing the maize ubiquitin promoter (up to 0.34% of total soluble leaf protein). GUS activities in transgenic in vitro-grown banana plants containing the My promoter were up to seven-fold stronger in leaf tissue and up to four-fold stronger in root and corm tissue than in plants harbouring the maize ubiquitin promoter. The Cv promoter showed activities that were similar to the maize ubiquitin promoter in in vitro-grown banana plants, but was significantly reduced in larger glasshouse-grown plants. In transgenic in vitro-grown tobacco plants, the My promoter reached activities close to those of the 35S promoter of cauliflower mosaic virus (CaMV), while the Cv promoter was about half as active as the CaMV 35S promoter. The BSV promoters for pregenomic RNA represent useful tools for the high-level expression of foreign genes in transgenic monocots.

A polymerase chain reaction (PCR)-based restriction fragment-length polymorphism (RFLP) assay was developed that discriminates among the 4 mitotypes found in North, Central, and South American honey bee racial groups—eastern European (Apis mellifera ligustica Spinola, caucasica Gorbachev, and carnica Pollman), western European (A. m. mellifera Linnaeus), Egyptian (A. m. lamarckii Cockerell Lepeletier), and sub-Saharan African (A. m. scutellata). Before the development of this assay, 13% of southern Californian feral bees collected before the arrival of the Apis mellifera scutellata (Africanized) race were found to contain a non-European mitochondrial genotype that could not be distinguished from that of A. m. scutellata. DNA sequence analysis suggests the unusual mitotype to be that of A. m. lamarckii. An RFLP polymorphism was identified that distinguished this subspecies from all others present in North America. This polymorphism was not found in any of 96 bees collected primarily in Mexico and Central America. Thus, the Egyptian mitochondrial type is either absent or extremely rare in these regions. The PCR assay also distin- guishes A. m. lamarckii from 2 other north African racial types, A. m. intermissa Buttel-Reepen and A. m. sahariensis Baldensperger.

We propose an Africanized honey bee identification strategy using morphometrics and an improved polymerase chain reaction (PCR)-based mitotyping procedure that distinguishes between feral and commercial bees maternally descendent from 4 racial groups-Eastern European (Apis mellifera ligustica, caucasica, and carnica), Western European (A. m. mellifera), Egyptian (A. m. lamarckii), and other African origins. Mitochondrial genotype is highly correlated with morphology. Ninety-five percent of morphometrically determined Africanized feral colonies collected in Texas, Arizona, California, and Mexico also contained African mitochondria. Sixty-two percent of colonies from commercial or minimally managed apiaries in Mexico and Central America, with Africanized forewing lengths and 17% of colonies with intermediate forewing lengths, had African mitochondria. The strong correlation between non-European morphology and African mitotype, as well as the speed and accuracy of mitotype determination, suggest a 3-step Africanized bee identification procedure. This identification procedure first examines forewing length. Bees with lengths above a given threshold (9.12 mm) have a very high probability of being pure European in origin and are not examined further. Those bees with wing lengths below the threshold are subjected to mitochondrial analysis (mitotyping). Samples having African mitochondria are not examined further. Those bees with small forewing lengths, but European mitotypes, are then identified using detailed morphometric discriminant function analysis. By performing these steps in sequence, the number of bees requiring full morphometric analysis is reduced, saving time and improving the accuracy of Africanized honey bee identification.

In the brain of the honey bee, Apis mellifera, the radioligands [3H]-SCH23390 and [3H]-spiperone recognise D1- and D2-like receptors, respectively. In addition to being pharmacologically distinct and exhibiting significantly different expression profiles during the lifetime of the bee, [3H]-SCH23390- and [3H]-spiperone-binding sites differ markedly in their distribution within the brain. Estimates of [3H]-SCH23390-binding site density are highest in the somatal rind, whereas [3H]-spiperone-binding sites are most concentrated in the beta lobe neuropil of the mushroom bodies. Molecular cloning techniques have been used to identify two honey bee genes encoding dopamine receptor homologs. The first is the honey bee counterpart of a Drosophila D1-like dopamine receptor and is expressed in the mushroom bodies of both workers and drones. The second is related to D2-like dopamine receptors from vertebrates and is expressed in the brain of the bee, but the precise distribution of expression is not yet known.

The biogenic amine receptor genes constitute an ancient and highly divergent family within the larger superfamily of G-protein-coupled receptors. These receptors play a central role in modulating nerve cell activity and thus behaviour. Because the honey bee offers numerous advantages for behavioural studies we endeavoured to isolate as many members of this gene family as possible from the bee. We compared numerous approaches to gene isolation and found that PCR amplification from small subfractions of cDNA or genomic DNA libraries enabled us to isolate clones that are otherwise undetectable. In total we isolated seven biogenic amine receptor clones and identified five additional related sequences by low-stringency Southern hybridization. Two clones, AmBAR4 and AmBAR6, are 84% and 72% identical to the Drosophila 5-HT2 and D1b receptors, respectively, and probably represent orthologous genes. Phylogenetic analysis indicates that AmBAR5 clusters loosely with a variety of tyramine and octopamine receptors with which it shares

Background Next-generation sequencing technology is an important tool for the rapid, genome-wide identification of genetic variations. However, it is difficult to resolve the ‘signal’ of variations of interest and the ‘noise’ of stochastic sequencing and bioinformatic errors in the large datasets that are generated. We report a simple approach to identify regional linkage to a trait that requires only two pools of DNA to be sequenced from progeny of a defined genetic cross (i.e. bulk segregant analysis) at low coverage ( Results Progeny from defined genetic crosses of Tribolium castaneum (F4 and F19) segregating for the phosphine resistance trait were exposed to phosphine to select for the resistance trait while the remainders were left unexposed. Next generation sequencing was then carried out on the genomic DNA from each pool of selected and unselected insects from each generation. The reads were mapped against the annotated T. castaneum genome from NCBI (v3.0) and analysed for SNP variations. Since it is difficult to accurately call individual SNP frequencies when the depth of sequence coverage is low, variant frequencies were averaged across larger regions. Results from regional SNP frequency averaging identified two loci, tc_rph1 on chromosome 8 and tc_rph2 on chromosome 9, which together are responsible for high level resistance. Identification of the two loci was possible with only 5-7× average coverage of the genome per dataset. These loci were subsequently confirmed by direct SNP marker analysis and fine-scale mapping. Individually, homozygosity of tc_rph1 or tc_rph2 results in only weak resistance to phosphine (estimated at up to 1.5-2.5× and 3-5× respectively), whereas in combination they interact synergistically to provide a high-level resistance >200×. The tc_rph2 resistance allele resulted in a significant fitness cost relative to the wild type allele in unselected beetles over eighteen generations. Conclusion We have validated the technique of linkage mapping by low-coverage sequencing of progeny from a simple genetic cross. The approach relied on regional averaging of SNP frequencies and was used to successfully identify candidate gene loci for phosphine resistance in T. castaneum. This is a relatively simple and rapid approach to identifying genomic regions associated with traits in defined genetic crosses that does not require any specialised statistical analysis.